Multi-epitope peptide sequence in-silico construction from HGV genome

In this study I have approached through in-silico method or reverse vaccinology taking advantage of the genome sequence of hepatitis G virus. It serves its benet of identifying antigens seen by both conventional as well as discovering any novel antigen. This peptide candidate can serve a triple purpose of hepatitis C vaccine, hepatitis G vaccine and HIV management addition. 89.2% of the residues were in the favoured region of Ramachandran plot. These points make it favourable for in-vitro trials and further renement. Because of the high similarity of hepatitis C genome to hepatitis G genome, it is highly probable that this peptide sequence might act as both hepatitis C and hepatitis G vaccine. Patients with past or current HGV infection have higher CD4+ lymphocyte counts and better AIDS-free survival rates. This peptide sequence might cause a breakthrough in the treatment of HIV without exposing them to develop hepatitis.


Introduction
In this study I have approached through in-silico method or reverse vaccinology taking advantage of the genome sequence of hepatitis G virus. It serves its bene t of identifying antigens seen by both conventional as well as discovering any novel antigen [1]. With technological advancement in the eld of immunology these studies have become easier and more accurate [2]. This peptide candidate can serve a triple purpose of hepatitis C vaccine, hepatitis G vaccine and HIV management addition.

Methodology
The procedure used in this study is entirely based on two previous studies [3] [4]. It will not be repeated here but is summarised below in gure 1 after obtaining necessary permission from its authors.
The modi cations done in this study is as follows: The genome sequence of hepatitis G virus was taken from NCBI virus database [5] (accession number:NC001710) Polyprotein precursor protein of this genome is used in this study. Docking of nal peptide sequence was done with tertiary structure of toll-like receptor 7 obtained from Protein Data Bank [6] (PDB ID:5GMF).

Result
The nal multi-epitope sequence formed after performing step 1 and 2 of methodology comprised of 214 amino acids: The above sequence distribution is as follows: The antigenicity prediction as in step 3 by Vaxijen server predicted it to be a probable antigen with score 0.462. The allergenicity prediction as in step 3 by Algpred server predicted it to be a non-allergen with score of -0.86908598 (positive predictive value is 0% and negative predictive value is 0%).
Physio-chemical properties as estimated by step 4 using ProtParam server gave the following results: The secondary structure of the nal multi-epitope sequence was computed using PHYRE 2 server: 40% comprised of alpha-helix, 3% of beta-strand, 13% of transmembrane helix and 22% was disordered ( Figure 2).
The tertiary structure obtained from PHYRE2 server was subjected to re nement by Galaxy Re ne tool which generated 5 models as follows (  The predicted B-cell linear epitopes were calculated using Ellipro suite ( Figure 5 and 6).
Toll-like receptor 7 was docked with the nal model by PatchDock server and top 10 results were re ned using FireDock server. Solution number 2 was the most favourable binding conformation with global energy at -4.41 and 0.00 repulsive Vander Waal forces. The docked model was visualized using UCSF Chimera (Figure 7).

Discussion
The protein sequence is predicted to be antigenic as well as non-allergic, hence proving its advantage of not producing any harmful hypersensitivity reaction in the body. It is basic in nature and has low molecular weight hence suitable for any route of administration except oral. It's half-life in E. coli is >10 hours, hence can easily be cultured and extracted. It is thermally stable as indicated by instability index. It has various B-cell epitope stimulating site and molecular docking with toll like receptor TLR-7 shows that it binds easily it without any repulsive Van der Waal forces. Toll-like receptor 7 which induces immune response against ss-RNA organisms will elicit an immune response against this sequence considering it be an active virus and thus ful lling its purpose as a vaccine. 89.2% of the residues were in the favoured region of Ramachandran plot. These points make it favourable for in-vitro trials and further re nement. All these studies were on web-tool prediction servers designed for such type of studies. Because of the high similarity of hepatitis C genome to hepatitis G genome, it is highly probable that this peptide sequence might act as both hepatitis C and hepatitis G vaccine [8]. Patients with past or current HGV infection have higher CD4+ lymphocyte counts and better AIDS-free survival rates [9][10] [11]. This peptide sequence might cause a breakthrough in the treatment of HIV without exposing them to develop hepatitis. However, since they work on growing databases, these cannot give a complete surety for success in future stages. Along with the advantage of the study, there are some limitations. 22% of the predicted secondary structure is disordered. Instead of 98% proteins being in the favourable region of Ramachandran plot only 89.2% of them is present. Advanced molecular dynamic simulations were not performed like RMSD (root mean square deviation). These disadvantages need to be overcome with better resources but these early results do serve as a guiding path to build future work upon it.

Conclusion
This study has highlighted a potential candidate ful lling its purpose as hepatitis C vaccine, hepatitis G vaccine and HIV management addition. In-depth studies and re nement might serve to be successful since it has very good results at such an early stage. It is needed to be validated experimentally.

Declarations
Con ict of interest: The author declares no con ict of interest.

Source of funding:
Nil.
Ethical consideration Not required. Epitope score chart from Ellipro suite Peptide model(red) docked with Toll-like receptor 7(blue).